Sandra Pellegrini

A protein tyrosine kinase in the interferon α β signaling pathway

The mutant human cell line 11.1 is unresponsive to interferon alpha. Here we describe the genetic complementation of this mutant and the identification and cloning of the wild-type gene that corrects the defect. Using transfection with genomic DNA in conjunction with a powerful back-selection, we isolated a cosmid that reverts the mutant phenotype of 11.1 cells. The cosmid encodes a single message whose level is greatly reduced in mutant cells. Complementary DNAs were cloned and found to be virtually identical to tyk2, a human mRNA encoding a non-receptor protein tyrosine kinase of previously unknown function. This finding shows that tyk2 links the interferon alpha/beta receptor to the cytoplasmic transcription factor that mediates activation of interferon-responsive genes.

Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.

Evolutionary dynamics of human Toll-like receptors and their different contributions to host defense.

Infectious diseases have been paramount among the threats to health and survival throughout human evolutionary history. Natural selection is therefore expected to act strongly on host defense genes, particularly on innate immunity genes whose products mediate the direct interaction between the host and the microbial environment. In insects and mammals, the Toll-like receptors (TLRs) appear to play a major role in initiating innate immune responses against microbes. In humans, however, it has been speculated that the set of TLRs could be redundant for protective immunity. We investigated how natural selection has acted upon human TLRs, as an approach to assess their level of biological redundancy. We sequenced the ten human TLRs in a panel of 158 individuals from various populations worldwide and found that the intracellular TLRs—activated by nucleic acids and particularly specialized in viral recognition—have evolved under strong purifying selection, indicating their essential non-redundant role in host survival. Conversely, the selective constraints on the TLRs expressed on the cell surface—activated by compounds other than nucleic acids—have been much more relaxed, with higher rates of damaging nonsynonymous and stop mutations tolerated, suggesting their higher redundancy. Finally, we tested whether TLRs have experienced spatially-varying selection in human populations and found that the region encompassing TLR10-TLR1-TLR6 has been the target of recent positive selection among non-Africans. Our findings indicate that the different TLRs differ in their immunological redundancy, reflecting their distinct contributions to host defense. The insights gained in this study foster new hypotheses to be tested in clinical and epidemiological genetics of infectious disease.

The Receptor of the Type I Interferon Family

All type I IFNs act through a single cell surface receptor composed of the IFNAR1 and IFNAR2 subunits and two associated cytoplasmic tyrosine kinases of the Janus family, Tyk2 and Jak1. A central issue in type I IFN biology is to understand how a multitude of subtypes can generate similar signaling outputs but also govern specific cellular responses. This review summarizes results from the last decade that contributed to our current state of knowledge of IFN-receptor complex structure and assembly.

The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression.

The four mammalian Jak tyrosine kinases are non‐covalently associated with cell surface receptors binding helical bundled cytokines. In the type I interferon receptor, Tyk2 associates with the IFNAR1 receptor subunit and positively influences ligand binding to the receptor complex. Here, we report that Tyk2 is essential for stable cell surface expression of IFNAR1. In the absence of Tyk2, mature IFNAR1 is weakly expressed on the cell surface. Rather, it is localized into a perinuclear endosomal compartment which overlaps with that of recycling transferrin receptors and with early endosomal antigen‐1 (EEA1) positive vesicles. Conversely, co‐expressed Tyk2 greatly enhances surface IFNAR1 expression. Importantly, we demonstrate that Tyk2 slows down IFNAR1 degradation and that this is due, at least in part, to inhibition of IFNAR1 endocytosis. In addition, Tyk2 induces plasma membrane relocalization of the R2 subunit of the interleukin‐10 receptor. These results reveal a novel function of a Jak protein on internalization of a correctly processed cytokine receptor. This function is distinct from the previously reported effect of other Jak proteins on receptor exit from the endoplasmic reticulum.

Early events in signalling by interferons

The heterogeneity of the biological responses to alpha/beta and gamma interferons, the degree of overlap, synergy and antagonism appear to reflect functional interactions between these two signalling pathways. Recent biochemical and genetic experimental approaches have identified some of the effector proteins involved in interferon signalling, and have advanced our understanding of the crosstalk between these signalling networks.

Interferon-α-dependent Activation of Tyk2 Requires Phosphorylation of Positive Regulatory Tyrosines by Another Kinase

Tyk2 and JAK1, members of the Janus kinase (JAK) family of protein tyrosine kinases, are required for interferon-α/β binding and signaling. Both enzymes are associated with the interferon-α/β receptor, and upon ligand binding, they undergo tyrosine phosphorylation and catalytic activation in an interdependent manner. To identify residues involved in Tyk2 regulation and to understand the basis of the interdependence of Tyk2 and JAK1, six mutated versions of Tyk2 bearing single or multiple point mutations in the tyrosine kinase domain were studied in a cell line lacking endogenous Tyk2. The Y1054F/Y1055F substitutions in the putative activation loop prevented ligand-dependent activation of Tyk2, without abolishing its catalytic potential. The K930R mutation in the ATP binding site generated a kinase-negative protein, which however, still became phosphorylated upon interferon-α treatment. The Y1054F/Y1055F substitutions in this kinase-negative Tyk2 abolished the induced phosphorylation. These results indicate that Tyk2 is activated by phosphorylation on Tyr-1054 and/or Tyr-1055 and that this phosphorylation requires another kinase, most likely JAK1. While the Tyk2 forms mutated on Tyr-1054 and Tyr-1055 or on Lys-930 allowed some inducible gene expression, the combination of the three point mutations totally abolished signaling.

Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons.

Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response to IFN-gamma was partially defective. Although many genes did respond to IFN-gamma in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN-gamma was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-alpha (E and M) or IFN-gamma (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN-gamma but not IFN-alpha. Band shift complementation assays revealed that untreated and IFN-gamma-treated U2 cells lack the functional E gamma subunit of transcription factor E and that IFN-alpha-treated U2 cells do contain the functional E alpha subunit.

Selective expression of type I IFN genes in human dendritic cells infected with Mycobacterium tuberculosis.

Type I IFN regulates different aspects of the immune response, inducing a cell-mediated immunity. We have recently shown that the infection of dendritic cells (DC) with Mycobacterium tuberculosis (Mtb) induces IFN-α. In this work we have monitored a rapid induction of IFN-β followed by the delayed production of the IFN-α1 and/or -α13 subtypes. The Mtb infection rapidly activates the NF-κB complex and stimulates the phosphorylation of IFN regulatory factor (IRF)-3, events known to induce IFN-β expression in viral infection. In turn, the autocrine production of IFN-β induces the IFN-stimulated genes that contain binding sites for activated STATs in their promoters. Among the IFN-stimulated genes induced in DC through STAT activation are IRF-1 and IRF-7. The expression of IRF-1 appears to be dependent on the sequential activation of NF-κB and STAT-1. Once expressed, IRF-1 may further stimulate the transcription of IFN-β. Induction of IRF-7 is also regulated at the transcriptional level through the binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears to be required for the delayed induction of the IFN-α1/13 genes. Although correlative, our results strongly support the existence of a cascade of molecular events in Mtb-infected DC. Upon infection, constitutively expressed NF-κB and IRF-3 are activated and likely contribute to the rapid IFN-β expression. In turn, IFN-β-induced IRF-1 and IRF-7 may cooperate toward induction of IFN-α1/13 if infection persists and these factors are activated.

The Janus kinase family of protein tyrosine kinases and their role in signaling

Abstract. In the early 1990s, the search for protein kinases led to the discovery of a novel family of non-receptor tyrosine kinases, the Janus kinases or JAKs. These proteins were unusual because they contained two kinase homology domains and no other known signaling modules. It soon became clear that these were not ‘just another’ type of kinase. Their ability to complement mutant cells insensitive to interferons and to be activated by a variety of cytokines demonstrated their central signaling function. Now, as we approach the end of the decade, it is evident from biochemical studies to knockout mice that JAKs play non-redundant functions in development, differentiation, and host defense mechanisms. Here, recent progress is reviewed, with particular emphasis on structure-function studies aimed at revealing how this family of tyrosine kinases is regulated.